ExtracellularDeterminantsofAnionDiscriminationofthe Cl ... · theNO 3 /H antiporter.SubstitutionoftheWTSerwithaPro is sufficient to convert CLC-5 from a Cl /H toaNO 3 /H antiporter

Extracellular Determinants of Anion Discrimination of theCl�/H� Antiporter Protein CLC-5*□S

Received for publication, June 17, 2011, and in revised form, August 18, 2011 Published, JBC Papers in Press, September 15, 2011, DOI 10.1074/jbc.M111.272815

Silvia De Stefano, Michael Pusch, and Giovanni Zifarelli1

From the Istituto di Biofisica, CNR, Via De Marini 6, I-16149 Genova, Italy

Background: The mechanism of anion selectivity of Cl� transporting CLC proteins is not fully understood.Results: Mutational analysis of the extracellularly exposed CLC-5 residue Lys210 indicates its essential role in aniondiscrimination.Conclusion:A positive charge at position 210 is crucial to confer NO3

� over Cl� preference in coupled anion/proton transport.Significance: These findings suggest a novel mechanism responsible for anion discrimination in CLC proteins.

Mammalian CLC proteins comprise both Cl� channels andCl�/H� antiporters that carry out fundamental physiologicaltasks by transporting Cl� across plasma membrane and intra-cellular compartments. The NO3

� over Cl� preference of a plantCLC transporter has been pinpointed to a conserved serine res-idue located at Scen and it is generally assumed that the other twobinding sites of CLCs, Sext and Sin, do not substantially contrib-ute to anion selectivity. Here we show for the Cl�/H� antiporterCLC-5 that the conserved and extracellularly exposed Lys210

residue is critical to determine the anion specificity for trans-port activity. In particular, mutations that neutralize or invertthe charge at this position reverse the NO3

� over Cl� preferenceof WT CLC-5 at a concentration of 100 mM, but do not modifythe coupling stoichiometry with H�. The importance of theelectrical charge is shown by chemical modification of K210Cwith positively charged cysteine-reactive compounds that rein-troduce the WT preference for Cl�. At saturating extracellularanion concentrations, neutralization of Lys210 is of little impacton the anion preference, suggesting an important role of Lys210

on the association rate of extracellular anions to Sext.

The central role of CLC proteins deduced by studies inmammals is the transport of Cl� ions across the plasmamembrane and intracellular compartments (1–4). This iscritical for muscle excitability, transepithelial transport,endocytosis, lysosomal function, and synaptic activity asproved by the involvement of several CLC human isoformsin hereditary diseases (5, 6).Interestingly, CLC proteins include both Cl� channels and

Cl�/H� antiporters. In the channels, Cl� ions passively diffusedown their electrochemical gradient, whereas in the transport-ers Cl� andH� are coupled with a stoichiometry of 2 Cl�:1 H�

(7–9). Despite the different thermodynamic transport mecha-

nisms, the overall architecture is conserved across the proteinfamily (10–14). Structures of the bacterial and eukaryotichomologues CLC-ec1 (11) and CLC-Cm (15) showed that theprotein assembles as a homodimerwith each subunit harboringa narrow permeation pathway containing 3 anion binding sites(Fig. 1, A and B). At the innermost binding site (Sin)2 the anionis still partially exposed to the intracellular solution. The centralbinding site (Scen) is located approximately halfway across themembrane, and ismostly contributed by conserved Ser andTyrresidues. The third and outermost binding site (Sext) is presentin the structure of CLC-Cm in which the side chain of a criticalglutamate residue, Gluext (Glu148 in CLC-ec1, Glu210 in CLC-Cm, andGlu211 in CLC-5), occupies Scen and in the structure ofCLC-ec1 when Gluext is neutralized (11). In WT CLC-ec1 theside chain of Glu148 occupies Sext and isolates the anion at Scenfrom the extracellular space. In the E148Q mutant, an anionoccupies Sext, whereas the Gln side chain is displaced from thepore (Fig. 1B). Gluext is crucial for gating and in CLC channelsits mutations induce a constitutively open phenotype (11, 12,16, 17). In the transporters Gluext is required for anion/protoncoupling and its substitution transforms the antiporters intopermanently open and pH independent anionic conductances(9, 18, 19).The three anion binding sites provide a structural framework

to understand the mechanism of anion selectivity of CLC pro-teins. Interestingly, the plant AtCLC-a is not a Cl�/H�

exchanger but rather a coupled 2NO3�:1 H� antiporter respon-

sible for NO3� accumulation into the vacuoles (20). The differ-

ent anion selectivity of AtCLC-a is surprising because poly-atomic anions like NO3

� and SCN� lead to uncoupling of anionand H� flux in CLC-ec1 and CLC-5 probably by slippage (18,21).The molecular determinant responsible for the different

selectivity forNO3� in coupled proton antiport activity has been

pinpointed to a single residue in the central binding site. Theconserved serine residue present in Cl�/H� antiporters (Ser107in CLC-ec1, Ser168 in CLC-5) (Fig. 1B) corresponds to a Pro in* This work was supported by Telethon Italy Grant GGP08064, the Italian Insti-

tute of Technology (Progetto SEED), and the Compagnia San Paolo.Author’s Choice—Final version full access.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. S1–S9.

1 To whom correspondence should be addressed: Istituto di Biofisica, CNR,Via De Marini 6, I-16149 Genova, Italy. Fax: 39-0106475500; E-mail: [email protected].

2 The abbreviations used are: Sin, internal binding site; Scen, central binding site;Sext, external binding site; MTS, methanethiosulfonate; MTSEH, 2-hydroxy-ethyl methanethiosulfonate; MTSES, 2-sulfonatoethyl methanethiosulfonate;MTSET, 2-(trimethylammonium)ethyl methanethiosulfonate; ITC, isothermalcalorimetry.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 51, pp. 44134 –44144, December 23, 2011Author’s Choice © 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

44134 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 51 • DECEMBER 23, 2011

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the NO3�/H� antiporter. Substitution of theWT Ser with a Pro

is sufficient to convert CLC-5 from a Cl�/H� to a NO3�/H�

antiporter and confers higher NO3� permeability to CLC-ec1

and CLC-0 (7, 22, 23), whereas the opposite substitutionendowedAtCLC-a with coupled Cl�/H� exchange activity (23,24). Consistent with an important role of Scen in the couplingmechanism, structural analysis showed that anion occupancyof the central binding site ofCLC-ec1 correlateswith the degreeof coupling with H� countertransport (21, 25).

These findings clearly indicate that the central binding site iscritical for anion selectivity and anion/H� coupling efficiency.However, the question remains as to whether other proteinregions are also involved in anion selectivity.Several lines of evidence obtained fromCLC-ec1 suggest that

the internal binding site is not a likely candidate. The fact thatanions at this position are still partially hydrated argues againsta role in anion selectivity (10, 11). Consistent with this, it hasbeen suggested that Cl� binding to Sin is weak (22) and that Sinis poorly selective between Cl� and SCN� (21).

The external binding site is in principle another site poten-tially involved in anion selectivity but its role has been investi-gated only sporadically and importantly, for CLC transportersit has been probed only by mutating the conserved Gluext (18,19, 26, 27). From these studies it was concluded that Sext doesnot have a substantial role in anion selectivity (22, 28). How-ever, considering the complex role of Gluext, it is conceivablethat its substitutions, besides modifying Sext, have drasticeffects on general transport properties that can potentiallyhinder the detection of selectivity changes. Here we investigatethe role of the external binding site of CLC-5 in anion discrim-ination by a different approach.A conserved positive residue (Arg147 in CLC-ec1 and Lys210

in CLC-5, but Trp209 in CLC-Cm) is adjacent to the importantGluext (Fig. 1, A and B) and is part of a sequence stretch highly

conserved in CLCs from different organisms (Fig. 1C). Thestructure of the E148Q mutant of CLC-ec1 (11) shows thatArg147 is in close proximity to the outermost Cl� ion with itsside chain directed toward the extracellular space and its back-bone probably participating in anion coordination at Sext (Fig.1B). These elements potentially suggest a role for this residue inthe discrimination between transported anions, a hypothesisthat we tested on CLC-5 by substituting Lys210 with severalamino acids.Our results indicate that a positive charge at position 210 is

responsible for the NO3� over Cl� preference of WT CLC-5.

Detailed analysis of the anion concentration dependence oftransport suggests that Lys210 is primarily involved in regulat-ing the voltage-dependent association of anions from the extra-cellular medium.

EXPERIMENTAL PROCEDURES

Molecular Biology—Oocyteswere obtained by dissection andcollagenase treatment of ovaries from Xenopus laevis frogs.Mutations were introduced by recombinant PCR as previouslydescribed (29). All constructs were in the pTLN vector (30) andwere expressed by injecting 25–50 ng of cRNA transcribedfrom linearized cDNA from the AmbionmMessagemMachineSP6 kit. Oocytes were kept in a solution containing (in mM) 90NaCl, 10 HEPES, 2 KCl, 1 MgCl2, 1 CaCl2, pH 7.5, for 3–6 daysat 18 °C.Two-electrode Voltage Clamp—Two-electrode voltage clamp

was performed with a Turbotec 03 amplifier (npi, Tamm, Ger-many) and the custom acquisition programGePulse. Measure-ments were performed at room temperature (20–25 °C).Unless otherwise stated, the voltage protocol consisted of volt-age steps from 120 to �60 mV in decrements of 20 mV from aholding potential corresponding to the resting potential of theoocytes (between �15 and �30 mV, a range that is typical for

FIGURE 1. Location of residue Lys210 mapped on the structure of the CLC-ec1 mutant E148Q (PDB entry 1OTU). A, dimeric structure of CLC-ec1 viewedfrom the membrane plane (extracellular side above and cytoplasmic side below). The two subunits are shown in green and cyan. Residue Gln148 is colored in red,Arg147, corresponding to Lys210 of CLC-5, is shown in blue, and Ser107 in orange. Chloride anions bound to Sext, Scen, Sint are shown in magenta. B, expandedrepresentation of the anion permeation pathway for one of the subunits. The positions of the three binding sites are also indicated by horizontal dashed lines.C, alignment of the sequence stretch comprising Lys210 (shown in bold) for several CLC proteins.

Extracellular Determinants of Anion Discrimination of CLC-5

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CLC-5 expression). Measurements of stationary current wereperformedby applying voltage steps of 20ms to 80mVevery 2 s.The standard bath solution contained (in mM): 100 NaCl, 4MgCl2, 10 HEPES, pH 7.3. For measurements of anion selectiv-ity, NaCl was substituted with equimolar amounts of NaNO3,NaBr, NaSCN, or NaI.For some batches of oocytes Br� and NO3

� gave rise to smallcurrent increases in uninjected oocytes compared with Cl�.This small contribution did not affect the results obtained forthe WT and the mutants with the highest expression, K210A,K210C, K210R, andK210Q, but had to be considered for K210Eand K210H. On the other hand, in some batches, I� and SCN�

significantly increased the currents elicited in uninjectedoocytes. Therefore the results for I� and SCN� on all the con-structs were corrected for their effect on uninjected oocytes inthe following manner. The contribution of endogenous cur-rents in the different measuring solutions was estimated for atleast 3 noninjected oocytes. The average difference between thecurrent in the different anions and the current in Cl� was thensubtracted from themeasurements in oocytes of the samebatchexpressing CLC-5.Solutions with the methanethiosulfonate (MTS) reagents,

MTSEH (2-hydroxyethyl methanethiosulfonate), MTSES(2-sulfonatoethyl methanethiosulfonate), MTSET (2-(trimeth-ylammonium)ethyl methanethiosulfonate)), and with DTT(dithiothreitol) were freshly prepared the day of the experimentby dissolving in Cl� or NO3

� bath solutions the appropriateamount of substance from stock solutions stored at �80 or�20 °C. The final concentration was 1 mM for MTS reagents(unless otherwise stated) and 10 mM for DTT, pH 7.3, adjustedwith H2SO4 or NaOH.MTS reagents and DTTwere purchasedfrom Biotum (Biotium Inc., Hayward, CA).For measurements of [Cl�]ext dependence the extracellular

solution with the highest [Cl�] contained (in mM) 300 NaCl, 4MgSO4, 10 HEPES, pH 7.3. Lower [Cl�] was obtained by sub-stituting in a bath solution similar to the standard one (4 mM

MgSO4 instead of 4 mM MgCl2) equimolar amounts of NaClwith NaAsp.For a quantitative analysis of the anion concentration

dependencewe assumed a simple saturating behavior (in agree-ment with the data) described by the following equation,

I�anion� �Imaxanion[anion]

�K1/2anion � �anion��

(Eq. 1)

where anion can be chloride or nitrate, Imax is the current atsaturating [anion], and K1/2 the concentration of half-maximalcurrent. Thus, the dependence of the normalized currents on[Cl�]ext was fitted at each potential with Equation 2,

Inorm �I��Cl��

I�100 mM��

�Cl� � �K1/ 2Cl � 100 mM�

�K1/ 2Cl � �Cl�� 100 mM

(Eq. 2)

providing an estimate of K1/2Cl .

The dependence of the macroscopic currents on [NO3�]ext

was analyzed by normalizing to the currents in the presence of100 mM Cl�. Thus, these normalized currents were fitted withEquation 3,

Inorm �I��NO3��

I��Cl� � 100 mM��

ImaxNO3

ImaxCl

[NO3]

100 mM

�K1/ 2Cl � 100 mM�

�K1/ 2NO3 � �NO3��

(Eq. 3)

using the value of K1/2Cl obtained from the fit of Equation 2. The

fit of Equation 3 provides two important parameters: K1/2Cl , the

concentration of half-maximal current in NO3�, and Imax

NO3/ImaxCl ,

the ratio of currents in NO3� and Cl� in saturating concentra-

tions of both anions.The voltage dependence of K1/2 for Cl� and NO3

� was fittedwith a Woodhull model (31),

K1/ 2�V� � K�0� � exp�� zFV

RT � (Eq. 4)

where K(0) is the apparent binding constant for the anion at0 mV, z is the apparent electrical distance of a hypotheticalanion binding site, V is the voltage, F, R, and T have theirusual meanings (F, Faraday constant; R, gas constant; T,absolute temperature).Data analysis was performedwith a custom software (written

in Visual C��, Microsoft) and the Origin program (OriginLabCorporation). Data are presented as mean � S.E. unless other-wise stated. Statistical differenceswere assessed using Student’st test.Extracellular pH Measurements Using a pH-sensitive

Microelectrode—Proton transport was qualitatively measuredby monitoring the acidification of the extracellular solutionclose to the oocyte using a pH-sensitive microelectrode asdescribed previously (19). Briefly, a silanized microelectrodewas back-filled with a proton ionophore (Mixture B, Fluka) anda solution containing phosphate-buffered saline, and con-nected to a custom high-impedance amplifier. The electrodeswere routinely checked and responded consistentlywith a slopeof 57–61 mV/pH unit. The pH-sensitive microelectrode wasgently pushed onto the vitelline membrane without rupturingthe plasma membrane. Application of a series of pulses to apositive test potential under voltage-clamp induced acidifica-tion of the extracellular solution. Solutions contained 0.5 mM

HEPES buffer (pH 7.3).Determination of the Relative Coupling Efficiency—The

microelectrode method cannot be used to determine absolutecoupling coefficients but can provide an estimate of the relativecoupling efficiency of anion/proton exchange comparing mea-surements in the presence of different anions performed on thesame oocyte with the pH-sensitive microelectrode in the sameposition (7). We applied a train of defined length of activatingvoltage pulses and measured the final change in extracellularpH, �pH, and the net current during the voltage pulses, Inet, inthe presence of Cl� or NO3

� from the same oocyte. The currentis the sumof the proton current, IH and the anion current, Ianion,and the latter is equal to ranion IH, where ranion is the numberof anions transported for each proton.

Inet � IH � Ianion � �1 � ranion�IH (Eq. 5)

As a first approximation, the acidification is proportional to theproton current,




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�pH IH (Eq. 6)

and thus Equation 7.

Inet

�pH� f�1 � ranion� (Eq. 7)

The proportionality factor, f, depends on the distance of themicroelectrode from the oocyte surface, the local density ofmembrane expression, and other unknown factors. For thesame oocyte and microelectrode positions, however, f can beconsidered identical for Cl� and NO3

�. Therefore, the ratio ofthese quantities in NO3

� and Cl�, respectively, defining the rel-ative coupling efficiency, erel, as,

erel �Inet(NO3)

�pH(NO3)

�pH(Cl)

Inet(Cl)�

1 � rNO3

1 � rCl(Eq. 8)

is a measure of the relative efficiency of the Cl�/H� exchangecompared with the NO3

�/H� exchange. A large number of erelindicates that NO3

� transport is less coupled than Cl� trans-port, i.e. rNO3 � rCl.

RESULTS

Mutational Analysis—To test the possible role of Sext in sub-strate specificity of CLC-5 we mutated the conserved lysineresidue at position 210 to Ala, Cys, Glu, His, Gln, and Arg.

All mutants gave rise to measurable currents (Fig. 2A) that,taking into account the variability of protein expression inoocytes and possibly a different turnover rate, had a magnitudesimilar toWT, except K210E and K210H for which the currentmagnitude was reduced (Fig. 2, A and B).Comparison of the macroscopic currents (Fig. 2A) shows

that in general the behavior of the mutants is similar to WT.They display the strong outward rectification typical of WT(note the lack of current at negative voltages) (18, 26, 32) andthe characteristic quasi-instantaneous activation with an addi-tional, residual, slow component (Fig. 2A). The voltage depend-ence in the positive voltage range also appears very similar withthe surprising exception of the conservative mutation K210Rfor which the voltage dependence is decreased compared withWT (Fig. 2A). To obtain a more quantitative description of thevoltage dependence we estimated the degree of rectification bycalculating the ratio of the current amplitudes at 120 and 80mV. The results reported in Fig. 2C confirm that K210R is theonly mutant with substantially altered voltage dependence.To test whether the mutants have normal Cl�/H� exchange

activity, we assessed proton transport activity bymeasuring thedegree of extracellular acidification produced by the activationof the constructs in Cl� containing extracellular solution at 100mV and found that it is qualitatively very similar to WT (datanot shown). Importantly, control experiments on noninjected

FIGURE 2. Expression and rectification of the currents for WT CLC-5 and Lys210 mutants. A, representative current recordings for WT and the indicatedmutants. B, average of the ratio between the current of WT and the indicated mutants at 100 mV. In each batch of oocytes, the ratio was calculated by dividingthe average current of the mutants and the WT. Data were obtained for at least three batches (n � 7). Error bars are S.D. The average current of the WT was 5.7 �1.8 �A (n � 38). C, average values of the ratio of currents at 120 and 80 mV calculated for each individual oocyte for WT (Lys210) and the indicated mutants (thenumber of experiments is the same as in panel B, error bars are S.D.). The difference with the WT value is statistically significant for K210R (p 10�6) and K210Cand K210E (p 0.05).




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oocytes did not elicit any detectable proton transport activity(data not shown). In conclusion, despite some minor differ-ences, all mutants retained the basic transport properties ofWT CLC-5.We next asked whether the mutations affected the anion

specificity of CLC-5. The strong rectification of macroscopiccurrents of CLC-5 precludes an estimation of anion selectivitybased on measurements of the reversal potential, which is tra-ditionally the method of choice to investigate selectivity of ionchannels (33) and has been applied also to transporters thatmediate macroscopic current at both positive and negativepotentials, including CLC-ec1 (27). However, as detailed under“Discussion,” the selectivity of transport proteins can be mea-sured by differentmethods, in particular by comparing the cur-rent amplitude in different ionic conditions (33, 34). The vari-ous methods, assessing different thermodynamic properties,are not expected to provide the same results (34) and thereforegreat attention has to be taken in comparing the selectivityobtained with different techniques. In this work, the selectivityof WT and mutant CLC-5 was assessed in terms of the relativemacroscopic current that allows comparison of different anionsaccording to the relative efficiency with which they are trans-ported. To avoid confusion between this type of measurementand the selectivity obtained from permeability ratios (or othermethods), wewill refer to the anion discriminationmeasured inthis work as substrate specificity for transport activity.Supplemental Fig. S1 shows representative current record-

ings of an oocyte expressing WT CLC-5 in extracellular solu-tions containing different anions (note the different current

scale for SCN�). In agreement with previous reports (7, 32) wefound that Br� slightly decreases the currents compared withCl�, whereas the reduction produced by I� is much more pro-nounced. NO3

� and SCN� increase the currents (18). The ratioIanion/ICl� of the current magnitude in the presence of a partic-ular anion with the current in the presence of Cl� at 100 mV(for SCN� at 80 mV) and at a concentration of 100 mM, isshown in Fig. 3, B and C.In performing these experiments several problems have to be

taken into account. First of all, for SCN�, due to the largeincrease in current magnitude, the ratio ISCN�/ICl� was calcu-lated at 80 mV to avoid errors due to series resistance (Fig. 3C).Moreover, to compensate for possible drifts of the currents dur-ing the perfusionwith different anions,measurementswithCl�

solutionwere repeated after each anion tested. Finally, if endog-enous currents were present their contribution was subtracted(see “Experimental Procedures”).In agreementwith previous results (7, 32) the transport activ-

ity ofWT followed the substrate specificity sequence, SCN� �NO3

� � Cl� � Br� � I� (Fig. 3). Fig. 3B shows that with theexception of K210H none of the mutations significantlyaffected the relative specificity of Br� and I� compared withWT. However, considering the small effect we did not investi-gate this aspect in further detail. The effect of the mutations onrelative specificity of NO3

� was in contrast much more consis-tent and robust. In fact, with the exception of the conservativemutation K210R, all mutants decreased the relative specificityof NO3

� (Fig. 3, A and B).

FIGURE 3. Selectivity of WT CLC-5 and Lys210 mutants. A, representative current recordings in Cl� solution (black trace) and NO3� solution (red trace) at 100

mV for two oocytes expressing WT CLC-5 and the mutant K210A, respectively. The ratio INO3

�/ICl� was 1.61 for WT and 0.67 for K210A. Traces for K210A werescaled to obtain the same current level in Cl� solution for WT and K210A. B, average currents in Br�, NO3

�, and I� relative to Cl� at 100 mV (n � 7). The differencewith the WT value of INO3

�/ICl� is statistically significant for all the mutants (p 0.01), for IBr�/ICl� and IIodide�/ICl� is statistically significant only for K210H (p 0.05). C, average of the currents in SCN� relative to Cl� at 80 mV (n � 7). The difference with the WT value is statistically significant for K210A, K210E, and K210Q(p 0.05).




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At 100mV, transport activity ofWTCLC-5 is higher inNO3�

than in Cl� with INO3

�/ICl� � 1.6. In contrast, INO3-/ICl� is only1.1 for K210H, whereas K210A, K210E, K210C, and K210Qreverse the preference ofNO3

� andCl� (INO3

�/ICl� 1)with thestrongest effect produced by the mutant K210A (INO3

�/ICl� �0.7). The strong effect of K210A on the relative NO3

�/Cl� pref-erence is also illustrated in Fig. 3A,which shows representativemacroscopic currents in NO3

� and Cl� for WT and K210A.SCN� robustly increases the WT current compared with

Cl�, (ISCN�/ICl� at 80mV is 2.8). However, ISCN�/ICl� is stronglyreduced for K210A, K210C, and K210E and for K210Q trans-port activity is similar in the presence of SCN� and Cl� (Fig.3C). Themutations with the largest impact onNO3

� specificity,K210A, K210E, and K210Q, are the ones that also affect morestrongly SCN� specificity (Fig. 3, B and C). Considering thestrong effect of K210A onNO3

� specificity, we felt compelled toprovide additional evidence that this mutation preserved thetransport properties of the WT.First of all, we analyzed the dependence of macroscopic cur-

rents ofWT and K210A on [Cl�]ext. Fig. 4A shows the depend-ence on [Cl�]ext for WT and K210A at 100 mV. At a concen-tration of 3 mM currents are just above background, but raising[Cl�]ext leads to a progressive increase of currentmagnitude forbothWT and K210A. The data points follow aMichaelis-Men-ten behavior and the fit with Equation 1 allows estimating theapparent K1/2 for Cl� (Fig. 4A). K1/2 decreases with increasingvoltage (Fig. 4B), consistent with the idea that inward Cl�movement is favored at positive voltages. The K1/2 voltagedependence could be described by a Woodhull model (Equa-tion 4), with K(0) values of 1.1 and 0.9 M and values for theapparent electric distance z of 0.50 and 0.67, respectively, forWT and K210A. This result indicates that K210A produces atmost a small effect on Cl� transport further supporting thenotion that this mutant retains the basic properties typical ofthe WT.To gain some insight into themechanism responsible for the

decreased NO3� permeability of K210A, we investigated

whether for thismutantNO3� transport is uncoupled frompro-

ton flux as for WT, or if the change in selectivity was due to a

modified coupling with proton countertransport. To this aimwe compared proton transport activity of WT and K210A inCl� and NO3

� solutions. Fig. 5A shows representative experi-ments in which we measured the level of extracellular acidifi-cation produced by activation of WT and K210A at 80 mV inCl� andNO3

� solutions. They clearly show that like for theWT,NO3

� transport by mutant K210A is less coupled to proton fluxcompared with Cl� transport. To have a quantitative estimateof the coupling efficiency in Cl� and NO3

� we calculated theratio between the macroscopic current and the associated pHchange, I/�pH, for the two anions obtaining the relative cou-pling efficiency, erel (see “Experimental Procedures”). The val-ues of this parameter forWTandK210A at both 80 and 100mVare not significantly different (p� 0.05) (Fig. 5B). This indicatesthat the degree of uncoupling produced by NO3

� is similar inWT and K210A.In summary, so far, our experiments indicate that the inver-

sion of the NO3� and Cl� specificity observed for the mutant

K210A (at 100 mV and 100 mM extracellular anion concentra-tions) is not due to a substantial modification of the Cl� andNO3

�/proton couplingmechanismand it is not accompanied bychanges in the voltage dependence of Cl� currents, nor in themechanism of Cl� permeation compared with WT.Modification of theMutant K210C withMTS Reagents—The

change in NO3� specificity produced by substitution of Lys210

with residues of different charge and properties is quite surpris-ing. However, this result suggests that a positive charge at posi-tion 210 is critical to confer NO3

� over Cl� preference toCLC-5. This interpretation makes a strong prediction. If itwould be possible to modify K210C with MTS reagents of pos-itive charge, we should be able to restore the preference forNO3

� over Cl� of the WT that is inverted in this mutant.Application of the positively charged MTSET, at a concen-

tration of 1 mM, produced a marked increase of the stationarycurrent when applied in NO3

� solution for 2 min (Fig. 6A). Theincrease of stationary currentwas accompanied by an alterationof theNO3

�/Cl� specificity (assessed from the ratio INO3

�/ICl� at100 mV) as illustrated in Fig. 6, B and C. Before modification,K210C passes larger currents in Cl� than in NO3

� (Fig. 6B),

FIGURE 4. Dependence of WT and K210A on [Cl�]ext. A, dependence on [Cl�]ext of the normalized currents for WT (n � 5, filled squares) and K210A (n � 3, opencircles) at 100 mV (error bars are smaller than symbols). Normalization was performed with the current at 100 mM. Curves are fits to Equation 2, with K1/2 as thefitted parameter resulting in values of 71 � 6 and 116 � 10 mM for WT and K210A, respectively. B, voltage dependence of K1/2. Symbols are the same as panelA. Lines are fit with Equation 4 with K(0) and z as the fitted parameters resulting in values of 1064 � 27 and 0.67 � 0.09 mM for WT and 860 � 15 and 0.50 � 0.06mM for K210A, respectively.




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similar to K210A (Fig. 3B). Modification with 1 mM MTSETproduces an inversion of specificity (Fig. 6B) that becomes sim-ilar toWT (Fig. 3,A andB). Both the stationary current increaseand the inversion of selectivity produced by MTSET could bereversed by washout with 10 mM DTT for 10 min (Fig. 6B). Asimilar conclusion is suggested by I-V relationships recordedbefore and after MTSET application and after DTT washout(supplemental Fig. S2).Although MTSET had only a minor effect on the station-

ary current amplitude of K210C when applied in Cl� solu-tion (supplemental Fig. S3A) it induced the same inversion inselectivity upon application in either NO3

� or Cl� solutions(Fig. 6 and supplemental Fig. S3B) and in both cases did notproduce other major alterations of the current properties(supplemental Figs. S2 and S4). Importantly, MTSET modi-fication had no effect on WT as assessed from the stationarycurrent level (supplemental Fig. S5A), the anion specificity(supplemental Fig. S5B), and the general current properties(supplemental Fig. S6).Application of 1 mM of the neutral reagent MTSEH did not

have any effect on the stationary current level (Fig. 7A), onNO3

�/Cl� specificity (Fig. 7B), or on general current properties(supplemental Fig. S7) of K210C. Importantly, application ofMTSEH prevented subsequent modification by MTSET (Fig. 7and supplemental Fig. S7) proving that MTSEH reacted withK210C such that the cysteine residue was not available for sub-

sequentmodifications. Thereforemodification of K210Cwith aneutral charge does not alter the preference of K210C for Cl�over NO3

�.The negatively charged reagentMTSES evenwhen applied at

a concentration of 4 mM for 11 min did not have any effect onthe stationary current level, onNO3

�/Cl� specificity, or on gen-eral current properties (supplemental Figs. S8 and S9). How-ever, unlike MTSEH, MTSES was not able to react with K210Cas it did not prevent or alter the subsequent modification byMTSET (supplemental Figs. S8 and S9). These results supportthe essential role of the positive charge at position 210 forNO3

�/Cl� specificity.The Mechanism of Anion Preference—The method we used

to assess anion specificity of the Cl�/H� antiporter CLC-5 isbased on the relative current level (in other words the relativetransport rate) at 100mV in the presence of different anions (ata concentration of 100 mM) compared with Cl�. In these con-ditions, the anion specificity can be determined by both theanion accessibility to the protein interior and the turnover rateof the anions. However, considering that anion accessibilityshould be rate-limiting for transport only at subsaturating con-centrations, an analysis of the concentration dependence of thespecificity can potentially provide a manner to distinguishbetween the two contributions. To this aim, we tested the effectof [NO3

�]ext on the relative specificity of Cl� and NO3� on WT

and K210A.

FIGURE 5. Proton transport properties of WT and K210A. A, representative recordings of the extracellular acidification produced by the activation of WT andK210A. Horizontal gray bars indicate stimulation with trains of voltage pulses to 80 mV (400 ms duration) in Cl� (filled squares) and NO3

� (open circles) solutions.B, average coupling efficiency of Cl� and NO3

� with H� transport, erel (Equation 8), at 80 and 100 mV measured for WT (n � 4) and K210A (n � 6) (errors bars areS.D.). The difference is not statistically significant (p � 0.05).




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Fig. 8A shows the dependence ofWT andK210A currents on[NO3

�]ext at 120 mV. The values are normalized to the currentin 100 mM Clext� and were fitted with Equation 3 (see “Experi-mental Procedures”). Two important pieces of information can

be obtained from this analysis. First, as for Cl�, theK1/2 voltagedependence for NO3

� could be described by aWoodhull model(Equation 4), with K(0) values of 3.1 and 11.9 M and values forthe apparent electric distance z of 0.74 and 0.62, respectively,

FIGURE 6. MTSET modification of K210C. A, representative current recordings showing the effect on the stationary current level of 1 mM MTSET (applicationindicated by a black bar) and of the washout with 10 mM DTT (indicated by a gray bar). Both reagents were dissolved in measuring solution containing 100 mM

NO3�. Voltage protocol consisted of a 20-ms pulse to 80 mV applied every 2 s. After MTSET application the experiment was interrupted to record I-V relation-

ships in Cl� and NO3� solutions. The dashed line represents the 0 current level. B, representative current recordings in Cl� (solid trace) and NO3

� (dashed trace),at 100 mV from the same oocyte as shown in panel A (before and after MTSET application and after washout with DTT). In these conditions the value of INO3

�/ICl�

was, respectively, 0.89, 1.50, and 0.91. C, average value of INO3

�/ICl� before and after MTSET application (n � 15) and after DTT washout (n � 6). The increase ofINO3

�/ICl� after MTSET modification is statistically significant (p 10�6).

FIGURE 7. MTSEH modification of K210C. A, representative current recordings showing the effect of 1 mM MTSEH on the stationary current level followed byapplication of 1 mM MTSET. Both reagents were dissolved in measuring solution containing 100 mM NO3

�. Application of MTSEH is indicated by a gray bar andapplication of MTSET by a black bar. After MTSEH application the experiment was interrupted to record I-V relationships in Cl� and NO3

� solutions. The dashedline represents the 0 current level. B, representative current recordings in Cl� (solid trace) and NO3

� (dashed trace), at 100 mV from the same oocyte shown inpanel A (before and after MTSEH application and after application of MTSET). In these conditions the value of INO3

�/ICl� was, respectively, 0.91, 0.91, and 0.88.C, average value of INO3

�/ICl� before and after MTSEH application (n � 6) and after application of MTSET (n � 3). The differences of the values of INO3

�/ICl� in thesethree different conditions were not statistically significant (p � 0.8).




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for WT and K210A (Fig. 8B). K210A therefore has a signifi-cantly increased K1/2 for NO3

� compared with WT (values at120mV) (Fig. 8B). Second, from the fit with Equation 3 the ratiobetween the maximal current in NO3

� and Cl�, ImaxNO3-/Imax

Cl , wasfound to be 2.9 for WT, and 2.0 for mutant K210A. Unfortu-nately, it was not possible to apply anion concentrations higherthan 300mM as this quickly damaged the oocytes. This possiblyaffects our estimates of Imax

NO3-/ImaxCl . However, despite possible

errors in the exact values, this result strongly suggests that insaturating concentrations of NO3

�, when NO3� access is not

rate-limiting, the turnover in NO3� is higher than in Cl� for

both WT and K210A. According to this view the differencebetweenWTandK210A in the relative preference forNO3

� andCl� emerges only when the NO3

� concentration becomes rate-limiting for transport.

DISCUSSION

Substrate discrimination is one of the defining properties oftransport proteins. CLCCl� channels and the bacterial Cl�/H�

antiporter CLC-ec1 transport anions by intrinsically differentthermodynamicmechanisms but are characterized by the sameanion selectivity sequence SCN� � Cl� � Br� � NO3

� � I�,that could be estimated from measurements of permeabilityratios (i.e. measurements of the reversal potential of the cur-rents) (27, 35, 36).Due to the strong outward rectification, this method cannot

be applied to the mammalian Cl�/H� antiporter CLC-5.Therefore, in this work anion selectivity was estimated by com-paring the magnitude of macroscopic currents. With thisapproach, the selectivity is assessed in terms of the transportactivity, i.e. the turnover rate supported by the different anionscompared with Cl�. This method has been already applied toinvestigate substrate specificity of CLC exchangers and othertransporters (7, 26, 32, 37, 38) and is highly analogous to theassessment of selectivity of ion channels performed by mea-surements of conductance, a technique that has been widelyused. In fact, according to Hille (33), the Goldman-Hodgkin-Katz theory defines two empirical measures of selectivity, per-meability ratio andmacroscopic current. Thesemethods probedifferent thermodynamic properties and therefore are not

expected to generate the same results. Importantly, whereasboth types of measurements can be influenced by parameterslike ion concentration, the permeability ratio method tends tobe less sensitive to experimental conditions compared with theuse of macroscopic currents (34). For this reason, in assessingselectivity with the macroscopic current method, it is impor-tant to define the dependence of the current on the experimen-tal factors (most notably ion concentration). Conforming to thedefinition normally adopted for transporters and to avoid con-fusion with selectivity measured with permeability ratios, wedefined the selectivity obtained in this work as substratespecificity.In agreement with previous results (7, 32), we found that the

CLC-5 specificity sequence is SCN� � NO3� � Cl� � Br� �

I�. In particular, properties of the NO3� transport through

CLC exchangers has recently received revived attention withthe discovery that the plant AtCLC-a homologue is a 2NO3

�:1 H� antiporter characterized by a low permeability toCl� (anion selectivity assessed by reversal potential isNO3

� � I� � Br� � Cl�) (20). This is in contrast with theuncoupling produced by NO3

� in the mammalian CLC-5 andthe bacterial CLC-ec1 exchangers and showed that themechanism of anion selectivity and anion/H� couplingdeserved deeper scrutiny. The molecular determinant of thedifferent selectivity for Cl� and NO3

� in coupled proton anti-port activity has been identified in a single residue in Scen,indicating that this site is important for anion selectivity oftransport and for the coupling efficiency of anion flux withH� countertransport (7, 22–24). Currently it is assumed thatno other protein regions, in particular Sext, influence anionselectivity in CLC channels and transporters. This conclu-sion mostly derives from studies on CLC-ec1 for which,however, the role of Sext has been probed exclusively mutat-ing the conserved Gluext (Glu148) (27). Based on measure-ments of the shift of the reversal potential (9) and anionbinding affinity by isothermal calorimetry (22), it was gener-ally concluded that mutation E148A did not influence anionselectivity (9, 22, 28). However, we think that conclusions onthe anion selectivity based on comparison between WT and

FIGURE 8. Dependence of WT and K210A on [NO3�]ext and voltage. A, dependence on [NO3

�]ext of the normalized currents for WT (n � 7, filled squares) andK210A (n � 8, open circles) at 120 mV. Normalization was performed with the current in 100 mM Cl� at 120 mV. Solid and dashed curves are fits to Equation 3, withK1/2 and Imax

NO3�/ImaxCl� as the fitted parameters resulting in values of 171 � 26 and 372 � 74 for K1/2 and 2.9 � 0.2 and 2.0 � 0.2 mM for Imax

NO3�/ImaxCl� , for WT and K210A,

respectively. B, voltage dependence of K1/2. Symbols are the same as in panel A. Lines are fit with Equation 4 with K(0) and z as the fitted parameters resultingin values of 3.2 � 0.6 and 0.74 � 0.07 M for WT and 11.9 � 3.4 and 0.62 � 0.05 M for K210A, respectively.




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mutants of the critical Gluext should be interpreted with cau-tion for CLC-ec1 and CLC transporters in general for thefollowing reasons.First and most importantly, WT CLC-ec1 is a Cl�/H�

exchanger with a 2:1 stoichiometry for which NO3� and SCN�

induce slippage. Mutations of Gluext turn the transporter intopure anionic conductance with an intrinsically different trans-port mechanism.Moreover, measurements of binding affinity by isothermal

calorimetry (22) concur with structural data (11) in suggestingthat in equilibrium conditions, in WT CLC-ec1, only Scen issignificantly occupied by anions, whereas in mutant E148Aboth Scen and Sext present a stably bound anion. This suggestsrelevant differences in the transport mechanism in WT andE148A due to the altered occupancy of the different bindingsites as indicated also by the surprisingly decreased transportactivity of the E148A mutant compared with WT (39).Although some of the aspects just discussed might simply

reflect our limited understanding of the complicated mecha-nism linking anion binding affinity and permeation throughCLC proteins, they also indicate that in general, a direct com-parison between the selectivity of WT and mutation of Gluextcan be misleading, not only for CLC-ec1 but for all CLC trans-porters. Therefore, we believe that a different and less invasiveapproach could be beneficial to assess the role of Sext in anionselectivity. Motivated by this idea we investigated whethermutations of residues potentially contributing to Sext wouldaffect anion selectivity of CLC-5, whereas preserving generaltransport properties.Here, we found that a conserved lysine residue at position

210, probably contributing to anion coordination at Sext, has alimited impact on general transport properties of CLC-5 but iscritical for its anion specificity. The ability of CLC-5 to toleratemutations of this residue preserving function is quite uniqueamong the CLC proteins in which the corresponding residuehas been analyzed. The lysine corresponding to Lys210 of CLC-5has been mutated in the CLC channels CLC-0 (Lys165), CLC-1(Lys231), and CLC-Ka (Lys165) (17, 40–42). In CLC-0, theK165R mutation drastically altered the gating properties con-ferring a pronounced inwardly rectifying phenotype (40). TheK165C mutation did not yield functional channels, indicating,differently from our results, very strict requirements on theproperties of the residue at position 165 of CLC-0 to preservefunction. However, modification of K165C with positive MTSreagents MTSEA and 3-aminopropyl methanethiosulfate (butnot with MTSET) restored channel function and modified thegating properties similarly to the K165R mutation (41). In anycase, no specific conclusion could be drawn about the role ofthe charge of this residue on anion selectivity of CLC-0. ForCLC-1 it has been suggested that substitutions of Lys231,besides modifying the gating properties, affected anion selec-tivity (17). However, these conclusions were based onmeasure-ments of the membrane potential of cells expressing currentswith a very pronounced inward rectification, rendering theassessment of relative permeability to different anions practi-cally impossible. In CLC-Ka, mutant K165R strongly reducedcurrents, whereas mutation to Gln led to a complete loss-of-

function. Therefore also for CLC-Ka no information could begained about the role of Lys165 on anion selectivity (42).

Two independent lines of evidence support the role of Lys210in anion specificity of CLC-5. 1) Substitution of Lys210 with Ala,Cys, Glu, His, and Gln, but not with Arg, produce a strongdecrease (K210H) or an inversion (K210A, K210C, K210E, andK210Q) of the relativeCl�/NO3

� specificity comparedwithWT(assessed at 100mV and 100mM anion concentration) with thestrongest effect observed for K210A. SCN� transport activitywas also identical toWT for K210R but was strongly decreasedby other mutations with the strongest effect observed forK210Q, which is equally permeable to Cl� and SCN�. Impor-tantly, substitutions with the largest impact on NO3

� specificityare the same as the one with the biggest effect on SCN� speci-ficity. However, none of the mutations induced major altera-tions of the halide (i.e. Br� and I�) specificity compared withWT.2) Similar to K210A, the K210C mutant has a higher speci-

ficity for Cl� than for NO3�, but its modification by the posi-

tively chargedMTS reagent MTSET is able to restore the NO3�

over Cl� preference of theWT in a reversible manner, whereasmodification with the neutral MTSEH was not able to alter theanion preference of K210C. Thus, it appears that a positiveelectric charge at position 210 is critical for NO3

� over Cl�specificity of CLC-5.Several independent elements suggest that the general trans-

port properties of Lys210 mutants, in particular K210A, are verysimilar to WT. First of all we found that all the mutants ana-lyzed retain two basic properties ofWTCLC-5, the strong out-ward rectification of currents and a similar proton transportactivity in the presence of Cl�.In particular, for the mutant with the largest effect on NO3

�

selectivity, K210A, we additionally probed other transportproperties. The decreased permeability of K210A for NO3

� isnot due to a change in coupling stoichiometry with H� com-pared with WT. It seems therefore that Sext controls anionspecificity but does not impact anion coupling to H� transport.This is different from the effect of the S168P mutation, locatedat Scen, which increased NO3

� over Cl� selectivity but alsoendowed CLC-5 with coupled NO3

�/H� antiport activity (7). Itwould be interesting to check whether these features reflectgeneral properties of Sext and Scen or are due to a specific role ofresidues Lys210 and Ser168.Moreover, we compared another transport property of WT

and K210A, investigating for the first time the dependence ofCLC-5 activity on [Cl�]ext. The small difference in the values ofK(0) and z for WT and K210A further argues for an intactmechanismofCl� gating and permeation inK210A and furthersupport the notion that K210A specifically alters anion speci-ficity of CLC-5. It should be noted that the value of the K(0)obtained here for WT CLC-5 (1 M) is 1000 times higher thantheCl� binding constantsmeasured forCLC-ec1 by isothermalcalorimetry (22). However, considering that our estimate isbased on transport measurements (i.e. a nonequilibrium proc-ess) a direct comparison of the two measurements is not war-ranted. In fact, estimates of ion affinity based onmeasurementsof equilibrium and nonequilibrium processes provide resultsthat can differ by several orders of magnitude as illustrated by




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analysis of the BK K� channel performed by Neyton andMiller(43).Further insight into the mechanism of ion transport of

CLC-5 was obtained by the analysis of the concentrationdependence of the NO3

�/Cl� preference. In general, transportefficiency depends on both the access rate of the anions to theprotein interior and their subsequent translocation. These con-tributions are difficult to dissect. However, the dependence oftransport efficiency on extracellular anion concentrations pro-vides a way to distinguish between the two regimens as theanion access rate can be expected to be rate-limiting for trans-port only at subsaturating anion concentrations. Interestingly,the preference for Cl� over NO3

� introduced by the K210Amutation at anion concentrations of 100 mM was reversed insaturating conditions indicating that when NO3

� availability isnot rate-limiting, NO3

� is preferred to Cl� as a substrate in bothWT and K210A.In summary, we showed that neutralization of the conserved

positive charge at position Lys210 alters anion specificity ofCLC-5 for polyatomic anions (i.e.NO3

� and SCN�) but not forhalides (i.e. Br� and I�). A detailed analysis of the K210Amutant indicated that the altered preference for NO3

� over Cl�appears at subsaturatingNO3

� concentrations. Considering theextracellular localization of Lys210, this potentially suggests thatthe K210A mutation reduces NO3

� access to a binding sitelocated in the transmembrane electric field.In conclusion, we believe this is the first study to unveil a role

of Sext in the substrate specificity of CLC proteins. This isimportant to understand the molecular basis of anion discrim-ination in CLC proteins in general and is relevant to elucidatetheir role in physiological settings in which selection betweendifferent anions is critical; an example is provided by plants thatgrow in soils rich in both Cl� and NO3

� and need to strictlydiscriminate between them; plant CLC homologues have beenshown to play a major role in this process (24, 44).

Acknowledgments—We thank A. R. Murgia and F. Quartino forexcellent technical assistance.

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Silvia De Stefano, Michael Pusch and Giovanni ZifarelliProtein CLC-5

Antiporter+/H−Extracellular Determinants of Anion Discrimination of the Cl

doi: 10.1074/jbc.M111.272815 originally published online September 15, 20112011, 286:44134-44144.J. Biol. Chem.

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Documents

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